The antitumor agent ecteinascidin 743 (Et 743): Characterization of its covalent DNA adducts and its effect on DNA repair mechanisms.

摘要

Ecteinascidin 743 (Et 743), a natural product derived from the Caribbean tunicate Ecteinascidia turbinata, is a potent antitumor agent currently in phase II clinical trials. Et 743 binds in the minor groove of DNA forming covalent adducts by reacting with the N2 of guanine. Although DNA is considered to be the macromolecular receptor for Et 743, the precise mechanism by which Et 743 exerts its remarkable antitumor activity has not yet been elucidated. The aim of this study is to provide a rationale for the antitumor activity of Et 743 by studying its fundamental interactions with DNA at the molecular level.;First, DNA structural distortions induced by Et 743 were characterized using gel electrophoresis. Surprisingly, Et 743 bends DNA toward the major groove, a unique feature among DNA-interactive agents that occupy the minor groove. Second, in order to gain further insight into the molecular basis behind the apparent sequence selectivity of Et 743, the stability and structure of Et 743 adducts at different target sequences were determined. Based on this data, the overall stability of the Et 743-DNA adducts were found to be governed by the DNA target sequence where the inability of Et 743 to form optimum hydrogen bonding networks with its optimum recognition sites leads to the formation of an unstable adduct. In addition, the reaction of Et 743 with DNA is reversible and the rate of the reverse reaction is a function of the target sequence. Thirdly, to gain insight into the cellular processing of Et 743 adducts, the recognition and incision of these Et 743-DNA lesions by DNA repair enzymes were examined using UvrABC nuclease. The Et 743-DNA adducts are recognized and incised by these repair proteins; however, the pattern of recognition indicate that the less favored, and less stable Et 743 adducts are generally incised at a higher efficiency than the more favored, more stable adducts. Hence, DNA repair capacity is impaired at stable adducts. Consequently, lesions that evade repair incision can interfere with biological processes, ultimately causing cell death.;In conclusion, the results from this dissertation demonstrate that Et 743 differs from other DNA alkylating agents by its DNA structural effects and its biochemical consequences, providing insight into its unique profile of antitumor activity.